The present invention relates to genes for floral regulation of plants and to methods for controlling plant flowering by regulating the expression of said gene. The present invention also relates to transgenic plants whose flowering time is modified in comparison with wild type plants by regulating the expression of said gene and to methods for generating said transgenic plants.
In order to resolve the worldwide food problem, developing technology for increasing the yield of food using biotechnology has been desired. Grain, which is one of main crops, is seed of plants and some vegetables are fruit of plants. For productivity increase of these plants, floral regulation for controlling growth of plants is an important key technology. On the other hand, flowering inhibition of vegetables, whose vegetative organs such as leaves or roots are marketed, prevents vegetative organs from stopping their growth and often increases their productivity. In addition, for many crops the suitable cultivating places are limited because of their species specificity of hereditary flowering behavior depending on environment. Modification of these properties by flowering regulation can expand the suitable cultivating places.
In molecular genetic studies using model plants such as Arabidopsis thaliana and Antirrhinum majas, many genes involved in identity determination of floral meristems or morphogenesis of floral organs have been isolated. Among these genes LEAFY and APETALA-1 genes are known to be forcedly expressed in the host plant Arabidopsis or poplar when introduced into these plants, thereby flowering the plants earlier. Since these genes are not fundamentally involved in floral budding (the transition from vegetative growth to reproductive growth), the use of these genes alone cannot arbitrarily regulate flowering. If the function of these genes is inhibited, the shape of inflorescence is changed, which is obvious from the phenotype of the mutants, and flowering cannot be regulated.
The embryonic flower mutant of Arabidopsis, in which flowering occurs immediately after germination, is known (Sung et al. (1992), Science, vol.258: p1645-). In this mutant, the function of a gene that maintains vegetative growth for a certain period of time after germination is thought to be lost. The flowering of wild-type Arabidopsis is thought to be inhibited by the expression of this gene. Although the approximate location of this gene on the chromosome is reported (Yang et al. (1995), Dev. Biol., vol.169: p421-), the result is far from helping the isolation of the gene and the gene has not yet been isolated.
An objective of the present invention is to isolate a gene for floral regulation (flowering regulating gene) and to provide a transgenic plant into which the gene is introduced. If a fundamental gene that regulates flowering is isolated, flowering time can be freely controlled by artificially regulating this gene.
The present inventors have succeeded in isolating mutant Arabidopsis that exhibits flowering immediately after germination because the function of the flowering regulating gene is lost and in identifying a single gene, which was mutated, in a wide region of the chromosome and isolating it. Furthermore, the present inventors have confirmed that this gene has flowering inhibiting function by introducing the gene into Arabidopsis and expressing it. Based on these findings, the present inventors have completed the present invention.
Moreover, the present inventors have discovered that the flowering regulating gene isolated from any kind of plant by hybridization or PCR technique based on the sequence of Arabidopsis flowering regulating gene has the function that complements the mutation of the Arabidopsis super early flowering mutant, inhibits flowering, and induces normal differentiation of stems and leaves.
Thus, the present invention relates to novel flowering regulating genes that exist extensively in plants, proteins with flowering regulating activity encoded by said genes, transgenic plants in which the expression of said gene is modified, methods for generating these plants, and methods for controlling the flowering time of plants by regulating the expression of said genes. More specifically, the present invention relates to
(1) a DNA encoding a protein having flowering regulating activity, wherein said DNA selected from the group consisting of:
i) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 1;
ii) a DNA encoding a protein comprising the amino acid sequence substantially identical to that of SEQ ID NO: 1;
iii) a DNA hybridizing a DNA encoding the protein comprising the amino acid sequence of SEQ ID NO:1;
iv) a DNA encoding a protein comprising amino acid sequences showing 50% or more and 60% or more homology with amino acids 278 to 348 and 465 to 607, respectively, of the amino acid sequence of SEQ ID NO: 1;
v) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 8.
vi) a DNA encoding a protein comprising the amino acid sequence substantially identical to that of SEQ ID NO: 8.
vii) a DNA hybridizing a DNA encoding the protein comprising the amino acid sequence of SEQ ID NO: 8; and
viii) a DNA encoding a protein comprising amino acid sequences showing 50% or more and 60% or more homology with amino acids 282 to 352 and 450 to 592, respectively, of the amino acid sequence of SEQ ID NO: 8.a DNA encoding a protein having flowering regulating activity, wherein said protein comprises the amino acid sequence of SEQ ID NO: 1;
(2) the DNA of (1), wherein said DNA of i) comprises the coding region of the nucleotide sequence of SEQ ID NO: 2;
(3) the DNA of (1), wherein said DNA of v) comprises the coding region of the nucleotide sequence of SEQ ID NO: 9;
(4) the DNA of (1), encoding a protein having a zinc finger structure;
(5) a protein having flowering regulating activity, encoded by the DNA of (1);
(6) the protein of (5), comprising the amino acid sequence of SEQ ID NO: 1 or 8;
(7) a recombinant double-stranded DNA molecule comprising an expression cassette comprising the DNA of (1);
(8) a recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituent elements of i) to iii),
i) a promoter that can transcribe in plant cells,
ii) the DNA of (1) or a part of it fused to said promoter in sense or antisense direction, and selectively, and
iii) a signal involved in transcription termination of RNA molecules and polyadenylation, wherein the signal functions in plants;
(9) a transformant into which the recombinant double-stranded DNA molecule of (7) is introduced;
(10) a transgenic plant cell into which the recombinant double-stranded DNA molecule of (8) is introduced;
(11) a method for producing a protein of (5), wherein the method comprises
(a) cultivating a transformant of (9) and
(b) recovering a recombinant protein from said transformant or the culture supernatant of it;
(12) a transgenic plant comprising transgenic plant cells of (10);
(13) a method for producing a transgenic plant of (12), wherein said method comprises
(a) introducing the recombinant double-stranded DNA molecule of (8) into plant cells and
(b) regenerating said plant cells;
(14) a DNA encoding an antisense RNA complementary to a transcription product of a DNA of (1);
(15) a method for regulating the flowering time of a plant, wherein said method comprises introducing the whole or a part of a DNA of (1) or the whole or a part of a DNA of (14) into a plant and expressing it, thereby changing the activity of a flowering regulating protein; and
(16) an antibody that binds to a protein of (5).